The topography of the active site of acetylcholinesterase and the acetylcholine receptor from Torpedo californica has been examined with the aid of a mononitroxide analog (I) of decamethonium and symmetrical dinitroxide analogs (I) of decamethonium and symmetrical dinitroxide analogs (II, III) of decamethonium and hexamethonium. The I50 values for the inhibition of acetyl-cholinesterase by labels I-III were 0.25 micron M, 0.18 micron M and 3.3 micron M respectively. Electron spin resonance (ESR) measurements indicated that the decamethonium dinitroxide label II bound to acetylcholinesterase in an extended conformation. The ESR spectrum also contained two highly immobilized components suggesting that each quaternary nitrogen experienced a different environment. These findings have provided direct proof for a two-point attachment of decamethonium analogs to acetylcholinesterase involving the active site and a peripheral anionic site. Label I exhibited the highest affinity for the acetylcholine receptor from which it was quantitatively displaced by a stoichiometric amount of alpha-bungarotoxin. The affinity of I for the receptor increased threefold during a 30 minute incubation. These findings suggest that the desensitization phenomenon observed for nicotinic agonists may result from a time-dependent increase in the affinity of these agents for the receptor.